Methods of rapid ligation-independent cloning of DNA and uses thereof

Abstract

The present invention generally relates to improved methods of assembly of two or more DNA fragments, methods of rapid ligation-independent cloning, and kits for rapid ligation-independent cloning and their uses.

What is claimed is: 
     
       1. A method of cloning DNA using T4 DNA polymerase comprising:
 a) combining linear DNA fragments having terminal sequence homologies between 15 and 100 nucleotides; 
 b) generating single-stranded recesses by adding the T4 DNA polymerase to the linear DNA fragments; and 
 c) treating the T4 DNA polymerase with heat at a temperature between 50° C. and 95° C. for a time of 10 seconds. 
 
     
     
       2. The method according to  claim 1 , wherein steps a) through c) are completed in 1 minute. 
     
     
       3. The method according to  claim 1 , wherein steps a) through c) are completed in less than 3 minutes. 
     
     
       4. The method according to  claim 1 , wherein steps a) through c) are completed in less than 5 minutes. 
     
     
       5. A method of cloning DNA using T4 DNA polymerase comprising:
 a) combining linear DNA fragments having terminal sequence homologies between 15 and 100 nucleotides; 
 b) generating single-stranded recesses by adding the T4 DNA polymerase to the linear DNA fragments; and 
 c) treating the T4 DNA polymerase with heat at a temperature of 75° C. for 5 seconds. 
 
     
     
       6. A method of cloning DNA using T4 DNA polymerase comprising:
 a) combining linear DNA fragments having terminal sequence homologies between 15 and 100 nucleotides; 
 b) generating single-stranded recesses by adding the T4 DNA polymerase to the linear DNA fragments; and 
 c) treating the T4 DNA polymerase with heat at a temperature of 50° C. for a time of 1 minute.